Intact RNA of high purity is essential for generating cDNA for cloning applications.

Total RNA or mRNA can be used in the reverse transcription reaction. Total RNA is generally sufficient for cDNA synthesis reactions. However, if desired, mRNA can be easily obtained using a polyA Spin mRNA Isolation Kit (NEB #S1560) or Magnetic mRNA Isolation Kit (NEB #S1550).

The amount of RNA required for cDNA cloning depends on the abundance of the transcript-of-interest. In general 1 ng to 1 μg total RNA or 0.1 ng to 100 ng mRNA are recommended.